Abstract

Transketolase belongs to the family of thiamin diphosphate dependent enzymes. The aim of this study was to
establish a bacterial expression system for human transketolase in order to investigate the functional characteristics
of mammalian transketolases. The level of recombinant human enzyme expressed in Escherichia coli was modest.
Puri®cation of recombinant transketolase and separation from the E. coli enzyme has been greatly simpli®ed by
means of a non-cleavable hexa-histidine tag. The highest speci®c activity was 13.5 U/mg and the Km values were
0.2720.02 and 0.5120.05 mM for the substrates D-xylulose 5-phosphate and D-ribose 5-phosphate, respectively.
Binding of cofactors to the apoenzyme showed the expected hysteresis. Without preincubation, the Km values for
thiamin diphosphate and for Mg2+ were, respectively, 4.120.8 and 2.520.4 mM, but after 1 h of preincubation
these values were 85216 nM and 0.7420.23 mM. The kinetic constants are similar to those of the native enzyme
puri®ed from human erythrocytes. Despite the modest expression level the reported system is well suited to a variety
of functional studies.